CB1 cannabinoid receptor: cellular regulation and distribution in N18TG2 neuroblastoma cells.
In order to characterize cellular regulation of CB1 cannabinoid receptors, synthesis and turnover studies were performed. Metabolic labeling of N18TG2 cells with 35S-labeled amino acids was followed by immunoprecipitation from cell lysates using an affinity-purified antibody generated to the N-terminal 14-amino-acid segment of the CB1 receptor. During a 2 h labeling period, CB1 receptors were rapidly and constitutively synthesized (rate: 0.86%/min). The majority of newly synthesized CB1 cannabinoid receptors (70%) was degraded rapidly by a first-order process (t1/2=4.8 h). The remaining nascent receptors, which were degraded slowly (t1/2>24 h), may represent the pool of potentially functional receptors. Trypsin treatment of intact confluent cells, designed to cleave the extracellular antibody recognition site, did not alter the recovery of metabolically labeled immunoprecipitated CB1 receptors. This suggests that a large percentage of newly synthesized receptors was inaccessible to the protease and is probably intracellular. Immunocytochemistry revealed CB1 cannabinoid immunoreactivity both intracellularly and on the cell surface. Subcellular membrane fractions exhibited receptor binding activity on plasma membranes and nuclear-associated membranes. Only low-affinity binding was seen in the chromatin fraction. An hypothesis has been developed to explain these results and form the basis for future studies.
McIntosh
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Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104, USA. mcintosh@sluvca.slu.edu
Song
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Howlett
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D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D009447:Neuroblastoma; D051381:Rats; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug / Q000096:biosynthesis* / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013462:Sulfur Radioisotopes; D013997:Time Factors; D014407:Tumor Cells, Cultured
D043882:Receptors, Cannabinoid; D011955:Receptors, Drug; D013462:Sulfur Radioisotopes; D008715:Methionine; D003545:Cysteine